Novel N-methylsulfonyl-indole derivatives: biological activity and COX-2/5-LOX inhibitory effect with improved gastro protective profile and reduced cardio vascular risks

Abstract Three novel series of N-methylsulfonylindole derivatives 3a&b, 4a–e, and 5a–e were synthesised. Different biological activities of the synthesised compounds were studied. Antimicrobial activity showed that, compounds 4b, 4e and 5d had selective antibacterial activity against the Gram-negative bacteria, Salmonella enterica and/or E. coli. The anti-oxidant activity of the synthesised compounds was evaluated by DPPH radical scavenging activity. In vitro anti-inflammatory activity was estimated. Compounds 4d, 4e, 5b, and 5d showed the highest anti-inflammatory activity. The COX-1, COX-2 and 5-LOX inhibitory activities were measured using enzyme immune assay (EIA) kits. Due to the dual COX-2/5-LOX inhibitory activity of compound 5d, its cardiovascular profile was determined by measuring cardiac biomarkers (LDH, CK-MB, and Tn-I). Besides, the histopathological study of the heart muscle and stomach were examined for the most active COX-2 inhibitors 4e and 5d. Finally, a molecular modelling study and pharmacokinetic properties were obtained using different computational methods.


Introduction
Pathogens, damaged cells and irritants are harmful stimuli that lead the body to make a complex response known as inflammation 1,2 .
Inflammation is important for the tissue repair process, but in the case of its chronic form, it causes negative effects on the body. Anti-inflammatory agents aim to relieve inflammatory symptoms such as pain, redness, heat and swelling 3,4 .
Non-steroidal anti-inflammatory drugs (NSAIDs) are the most common method of treatment of inflammatory symptoms. They act through inhibition of biotransformation of arachidonic acid (AA), a membrane bound phospholipid, to prostaglandins (PGs), prostacyclines (PGI2) and thromboxane A2 (TXA2) by the action of cyclooxygenase (COX) enzymes (COX-1, 2, 3) [5][6][7] . COX-pathway inhibition leads to unwanted side effects such as ulcerogenicity, hepatic and renal toxicity, which have arisen due to COX-1 inhibitors and cardiovascular disorders caused by COX-2 inhibitors. Both NSAIDs that could inhibit both COX-1 and COX-2 enzymes, such as aspirin, phenazone and indomethacin, (Figure 1), as well as selective COX-2 inhibitors, especially like, roficoxib and valdecoxib, (Figure 1) can increase the cardiovascular risks specially in patients with pre-existing cardiovascular disease. This can be caused by the imbalance in PGI2 (potent vasodilator and antithrombotic)/TXA2 (prothrombotic) ratio. Consequently, most drugs have been used in a restricted manner or even withdrawn from the market [8][9][10] . This leads to a search for new compounds that act in another way to metabolise AA. Liopoxygenase (LOX) enzymes  convert AA to leucotrienes. 5-LOX is the one associated with inflammation, bronchoconstriction, allergy and asthma [11][12][13] .
As a result, designing new compounds with dual COX-2/5-LOX inhibitory activity might solve the problem by offering new options for developing anti-inflammatory agents with the advantages of selective COX-2 inhibition and at the same time have better cardioprotective profile 2,14,15 .
Several biomarkers were reported to be used for the assessment of heart function such as sera containing aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), troponin (Tn-I) and creatine kinase-MB (CK-MB) in addition to tumour necrosis factor-a (TNF-a), a key player in the inflammatory response and cardiac depression, interleukin-6 (IL-6) and interleukin-1b (IL-1b) as the decrease in their level indicates low risk of cardiovascular toxicity [16][17][18] .
Besides, tissue including glutathione (GSH) acts as an antioxidant in cardiac tissue. It was reported that chronic administration of celecoxib resulted in an increase in lipid peroxidation via elevation of oxidative stress markers, which in turn increases oxidative kidney damage, leading to a decrease in GSH level 19 .
Moreover, one of the most important issues related to inflammation is with bacterial infections, which can be considered a primary cause of inflammation area as well as a second complication due to the accumulation of fluid inside the injured area 20 .
Resistance is a serious medical problem that has been observed in the currently used antibacterial agents. It makes the treatment of infectious diseases very difficult. So, the discovery of novel antibacterial agents might be the best way to overcome this episode 21,22 .
Celecoxib, (Figure 1), the drug of choice as a COX-2 inhibitor, was investigated as an antimicrobial agent as well. It showed potent effects in the reversal of multidrug resistance in MRSA, besides the increased sensitivity of M. smegmatis and S. aureus to antibiotics 23 .
Another way that affects inflammation is the free radical formation which causes cell damage and inflammation. So, anti-oxidant agents are taken side by side with anti-inflammatory drugs in most cases 24,25 .
Searching the literature, it was found that indole is a nucleus of choice as a multi-target scaffold for the treatment of inflammation besides its antimicrobial and anti-oxidant activities [26][27][28][29] .
This lead scaffold is a universal constituent in pharmacologically active natural products as well as synthetic drugs 30 .
Thus, indole thiosemicarbazide derivatives showed both anticancer and anti-oxidant activity 26 , indole alkaloids obtained from Alstonia scholaris and Topsentia sponge showed antibacterial activity 4,31 . Indomethacin, the indole derivative, is considered one of the most promising synthetic drugs as an anti-inflammatory and analgesic agents 8 .
Taking into consideration the various reported biological activities associated with indole, carbazones, and thiazolidinone skeletons, it was worth incorporating these moieties together in a single frame "privileged medicinal scaffolds" to obtain a more potent and biologically active multi-target drug with fewer side effects and a high safety profile.
The design of our newly suggested compounds using the active multi-target drug strategy, (Figure 1), depends on; (i) the main scaffold is indole as indomethacin, the lead compound, (ii) COX-2 pharmacophore, SO 2 Me, from rofecoxib structure, (iii) electron donating substituent on p-position of the phenyl ring, to mimic celecoxib, the selective COX-2 inhibitor drug, (iv) introducing carbazone (semi/thiosemi) moieties with reported anti-inflammatory, anti-oxidant and antimicrobial activities on indole C-3, (v) merging thiazolidinone ring with indole scaffold comes from 5-LOX inhibitors drabufelone and tenidap, respectively 3 .
The structure and stereochemical configuration of three series of target compounds 3&b, 4a-e and 5a-e were confirmed. In vitro anti-inflammatory activities through the determination of TNF-a inhibition in RAW264.7 macrophages, COX-1/2 and 5-LOX inhibitory activities were evaluated. The gastrointestinal and cardiovascular evaluation was determined. The plausible binding interactions inside COX-2 and 5-LOX active sites were explored using a molecular modelling study. Moreover, anti-oxidant and antimicrobial activities were investigated. Finally, ADME prediction and drug-likeness parameters were investigated.

Chemistry
Melting points were determined using the Griffin apparatus and were uncorrected. Values of IR spectra were measured using Shimadzu IR-435 spectrophotometer with KBr discs and represented in cm À1 . 1 H NMR and 13 C NMR were carried out using the Bruker instrument at 400 MHz for 1 H NMR and 100 MHz for 13 C NMR spectrophotometer, (Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt), in DMSO-d6 (as a solvent), D 2 O using TMS as an internal standard and chemical shifts were recorded in ppm on the d scale using DMSO-d6 (2.5) as a solvent. Coupling constant (J) values were estimated at Hertz (Hz). Splitting patterns were designated as follows: s, singlet; d, doublet, t, triplet; q, quartette; m, multiplet. Hewlett Packard 5988 spectrometer (Palo Alto, CA) was used to record the electron impact (EI) mass spectra (Microanalytical centre, Cairo University). Microanalysis was performed for C, H, and N on Perkin-Elmer 2400 at the Microanalytical Centre, Cairo University, Egypt and was within ±0.4% of theoretical values. Analytical thin-layer chromatography (TLC), pre-coated plastic sheets, 0.2 mm silica gel with UV indicator (Macherey-Nagel) was employed routinely to follow the course of reactions and to check the purity of products. All other reagents, solvents and compound 1 were purchased from the Aldrich Chemical Company (Milwaukee, WI) and were used without further purification.
General method for preparation of compounds 3a&b and 4a-e A mixture of indole derivative 2 (2.23 g, 0.01 mol) and the appropriate semicarbazone or thiosemicarbazone derivative (0.01 mol) in absolute ethanol (20 ml) containing drops of DMF, was heated under reflux for 2-4 h. The obtained solid was filtered, dried, and crystallised from 95% of ethanol to give compounds 3a&b and 4a-e.

Biological evaluation
Antimicrobial activity screening All compounds were screened for their antimicrobial activity at the Microbiology and Immunology Department, Faculty of Pharmacy, Beni-Suef University. The antimicrobial activity screening was carried out using the disc diffusion method, as described earlier [36][37][38] , with some adjustments.
For each indicator microbe, a solution of half McFarland turbidity was prepared in sterile saline, followed by surface streaking of the strains on Mueller-Hinton agar with a sterile cotton swab. Then, sterile filter paper discs (5 mm diameter) preloaded with 100 mg of each compound were applied to the surface of the preinoculated plates. Then, the plates were chilled at 4 C for 100 min before being incubated overnight at the proper temperature for microbial growth. Finally, the diameter of the inhibition zones around each disc was measured to evaluate the antimicrobial activity of the compounds. Ciprofloxacin was used as a standard antibacterial agent at a concentration of 20 mg/disc. Furthermore, DMSO-loaded discs were used as a negative control.
The DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical assay was carried out according to the method of Boly et al. 2016 39 . Briefly, freshly prepared DPPH reagent (100 ml, 0.1% in methanol) was added to 100 ml of the sample on 96 well plates (n ¼ 6). The reaction was incubated at room temp for 30 min in the dark. After completion of the incubation time, the reduction in DPPH colour intensity was measured at 540 nm. Data obtained was represented as means ± SD, according to the following equation, percentage inhibition ¼ [(Average absorbance of blankaverage absorbance of the test)/(Average absorbance of blank)] Ã 100. The results were recorded using a microplate reader, FluoStar Omega.
Microsoft Excel V R was used to analyse the obtained results. The IC 50 values were calculated using Graph pad Prism 6 V R by converting the concentrations to their logarithmic value and selecting the nonlinear inhibitor regression equation (log (inhibitor) vs. normalised responsevariable slope equation). The methods used and the final test report (R-SO7320) were obtained from Nawah Scientific.

Anti-inflammatory activity
Measurements of TNF-a in RAW264.7 macrophage cells

Reagents and chemicals
Both Lipopolysaccharides (LPS) and hispidin were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) 40 . The foetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Hyclone (General Electric Healthcare Life Sciences, Mississauga, Canada), penicillin streptomycin (P/S) was purchased from Solarbio (Solarbio life sciences, Beijing, P. R. China). Results of all in vitro anti-inflammatory screening were obtained from the confirmatory diagnostic unit, VACSERA, Egypt.
Cell culture DMEM was used to cultivate the macrophage cells RAW264.7 (Shanghai BOGO Industrial Co., Ltd., Shanghai, China), and supplemented with 10% FBS and 1% P/S (100 U/ml and 100 mg/ml, respectively). The cells were maintained in DMEM at 37 C and 5% CO 2 . They were treated with various concentrations of hispidin, then with 1 lg/ml LPS for the indicated time.

Cell viability assay
The RAW264.7 macrophage cells were seeded into 96 well plates at a concentration of 4 Â 10 3 cells per well, with different concentrations (100 to 0.4 lg/ml) of hispidin treated 24 h. In the next step, to each well, a solution of 5 mg/ml 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich,) was added and incubated (37 C, 5% CO 2 ) for 4 h. After that, the supernatant was removed and DMSO was added to dissolve formazan. Finally, the absorbance was measured at 490 nm using a UV MAX and kinetic microplate reader (Molecular Devices, LLC).
Reverse transcription quantitative PCR (RT-qPCR) gene expression RT-qPCR analysis was performed in one-step SYBR Green RT-qPCR. Each reaction contained: 1 ml of cell lysate, iScript One-Step SYBR Green RT-PCR Supermix (170-8893; Bio-Rad), 600 nM of each primer, and nuclease-free water to 10 ml. A CFX96 real-time PCR instrument (Bio-Rad) was used under the following protocol: 50uC for 10 min, 95uC for 5 min, 95uC for 10 s/61uC for 15 s/72uC for 30 s.
After the 72 C extension step, data collection occurred. Total RNA purified from cells was used as an RT-qPCR quantification standard.
In vitro lipoxygenase (5-LOX) inhibition assay The ability of the test compounds to inhibit the 5-LOX enzyme (IC 50 value, lM) was detected using the Cayman Human Lipoxygenase Inhibitor Screening Assay (EIA) kit (catalog no. K980-100, Cayman Chemical). The IC 50 values of test compounds were measured in mM according to the manufacturer's instructions and reported method 43 .

Animal and ethics statement
In this study, adult male Wister albino rats weighing 160-180 g were used. Before any experimental procedure, rats were given 14 days to acclimate. These rats were maintained in a controlled setting with food and water available. All experiments were conducted in accordance with laboratory animal care guidelines. All experimental procedures were carried out in accordance with the regulations of the Committee of Ethics for Scientific Research on Living Organisms, Faculty of Pharmacy, Nahda University, Beni-Suef (NUB). The approval number was (NUB-019-025).

Ulcerogenic liability
Ulcerogenic susceptibility was assessed for the most selected candidates (5b and 4e) and compared to indomethacin using celecoxib as a standard. Before drug administration, rats were fasted for 18 h and then separated into four groups. The control group received a suspension of the vehicle (10% DMSO in saline was given P.O (100 mg/kg) once daily). Other groups were given candidates, celecoxib, and indomethacin, in accordance with the described methodology 44 . Rats were given the required dose for three consecutive days. Following the last dose, the stomachs of each rat were dissected and opened via the greater curvature and rinsed with saline. For investigation, the dissected stomach was stretched with pins on a cork board. Through illuminated magnifying lenses, the stomach mucosa was examined for the existence of ulcers. Cho and Ogle's method 45,46 was used to calculate the ulcer index.

Histopathological examination of stomach
Histopathological tests were performed to evaluate the effects of chemicals (5b and 4e) on the stomach on those of celecoxib and indomethacin, which served as a positive control. In brief, the stomachs of each animal group's rat were fixed in 10% neutral buffered formalin for 48 h. The sections were then dehydrated, cleared, fixed in paraffin, sectioned (5 m), and stained with H&E stain.

Cardiovascular evaluation
Eighteen rats were divided into three groups at random (6 animals in each group). The first group was given a vehicle and served as normal control. For two weeks, a suspension of the studied compound 5b and celecoxib in a 10% DMSO in saline was given P.O (100 mg/kg) once daily. Ketamine (100 mg/kg) was used to anaesthetise the animals on the 15th day. Blood samples were obtained from the retro orbital, allowed to clot, centrifuged at 1000 g for 15 min, and utilised to examine heart function biomarkers (LDH, CK-MB, and troponin). The abdomen of each rat was opened, and the heart was extracted and washed in ice-cold physiological saline before being harnessed for histological investigation.

Assessment of the cardiac function biomarkers
Troponin, LDH, and CK-MB activities were measured in rats' sera by spectrophotometer using commercial kits in accordance with manufacturers' instructions to assess the effect of compound 5d on cardiac function biomarkers in rat serum and then compared to celecoxib [47][48][49] .

Histopathological studies of heart
A histopathological examination was conducted to compare the effect of compound 5b on the heart to that of celecoxib. In brief, the hearts of rats given compound 5b and celecoxib, as a control, were fixed in 10% neutral buffered formalin for 48 h. The sections were then dehydrated, cleared, fixed in paraffin, sectioned (5 m), and stained with H&E stain 46,50 .

Molecular docking study
Molecular Operating Environment (MOE, 2014.0901) software was used in this study for docking analysis. The crystal structure of both COX-2 bound to its ligand celecoxib and 5-LOX with its ligand arachidonic acid were downloaded from the protein data bank (PDB: 3LN1, https://www.rcsb.org/structure/3LN1 and PDB: 3V99, https://www.rcsb.org/structure/3V99, respectively), with the resolution of 2.40 Å for COX-2 isozyme and 2.25 Å for 5-LOX enzyme. For two enzymes, the crystallised ligands were docked and the binding energy scores, amino acid interactions and relative mean square deviation (rmsd) were calculated. The well fitted and the least energetic poses were selected. The same docking protocol was operated for the synthesised compounds inside both COX-2 and 5-LOX active sites. For each docked compound, the most superposed conformer with the ligand with binding interactions resembling that of the ligand was chosen. The energy binding scores (Kcal/mol) and bond length values ( Å) were recorded. 2D and 3D pictures of the most predicted active compounds were taken.

Molecular properties and drug-likeness
Molinspiration (2018.02 version) 51 was used to calculate molecular properties such as molecular weight (MW), the number of hydrogen-bond acceptors (HBA), number of hydrogen-bond donors (HBD), partition coefficient (MlogP), number of rotable bonds (nrotb), topological polar surface area (TPSA), and Violation of Lipinski's rule of five (n-violation). Drug-likeness scores were also determined for all target compounds 3a&b, 4a-e and 5a-e.

Bioactivity prediction
Bioactivity properties of the target compounds 3a&b, 4a-e and 5a-e were checked. The obtained results of a G-protein coupled receptor (GPCR) ligand, ion channel modulator, a kinase inhibitor, nuclear receptor ligand, protease inhibitor, and enzyme inhibitor using molinspiration 51 were also recorded.

In silico ADME prediction
To predict in silico ADME properties of the synthesised compounds 3a&b, 4a-e and 5a-e. PreADME online surver 52 was used. Human intestinal absorption (HIA), cell permeability of CaCo-2 cell and Madin-Darby Canine Kidney (MDCK) cell, plasma protein binding (PPB), blood brain barrier (BBB) and skin permeability (SP) were calculated.

Metabolism prediction
The most important parameters used to measure metabolism and excretion were cytochrome P450 (CYP) isoforms. Metabolism prediction parameters for the tested compounds 3a&b, 4a-e and 5a-e were examined using the Swissadme online server 53 .

Statistical analysis
The obtained data was represented as means ± standard deviations (SD). Significant results were considered when Ã p˂0.05 or ÃÃ p˂0.005 using a student's t-test compared to reference drugs.
The obtained values were represented as a result of triple independent experiments.

Results and discussion
Chemistry General chemistry information The synthetic procedures for semicarbazone derivatives 3a&b, thiosemicarbazone derivatives 4a-e and thiazolidinone derivatives 5a-e were performed as depicted in Scheme 1.
Starting from indole-3-carboxaldehyde (1), N-methylsulfonylindole carboxaldehyde derivative 2 was prepared through an Nalkylation reaction using a strong base such as sodium hydride 54 . The synthesised key intermediate 2, reacted with semi/thiosemicarbazides in the presence of absolute ethanol containing a few drops of DMF to obtain semi/thiosemicarbazone derivatives 3a&b/ 4a-e via a condensation reaction. Afterward, the obtained thiosemicarbazone derivatives 4awere cyclized using ethyl chloroacetate and sodium acetate to provide the respective thiazolidinone derivatives 5a-e at good yields ranging from 68 to 79%.
To confirm the chemical structure of the synthesised compounds 3a&b, 4a-e, and 5a-e, 1 H NMR, 13 C NMR (DEPT-Q), mass spectral data and elemental analysis were determined.
IR spectra of compounds 3a&b, and 4a-e displayed stretching of NH and C ¼ O/or C ¼ S bonds at (3431-3122), (1696 and 1673), or (1129-1058) cm À1 , respectively. Additionally, 1 H NMR spectra for semi/thiosemicarbazone derivatives 3a&b/4a-e displayed signals at d 3.49-3.54 and 8.11-8.70 ppm corresponding to -SO 2 CH 3 and azomethine -CH ¼ N-protons, sequentially. Moreover, two D 2 O exchangeable singlet signals were observed at d 7.50-11.78 ppm attributed to NH protons of compounds 3a&b and 4a-e. 13 C NMR spectra of 3a&b and 4a-e showed peaks at d On the other hand, the structure of thiazolidinone derivatives 5a-e was confirmed using IR spectra through the appearance of an absorption band at 3429 cm À1 attributed to thiazolidinone NH in compound 5a and a band in the range of 1620-1715 cm À1 characteristic of C ¼ O in 5a-e. Additionally, the disappearance of the NH group of the parent compounds in all derivatives 5a-e confirmed the cyclisation process.
Another evidence for ring closure in compounds 5a-e was obtained from 1 H NMR spectra, in which a new singlet signal appeared at the range of d 3.93-4.12 ppm attributed to CH 2 protons, in addition to the disappearance of D 2 O exchangeable singlet signals for their parent compounds 4a-e, while, the appearance of thiazolidinone NH proton of compound 5a at d 7.51 ppm.
Similarly, the 13 C NMR spectra of 5a-e showed chemical shifts for carbons of CH 2 and C ¼ O groups in a region of d 32.59-33.53 ppm and d 172. .69 ppm, respectively. Additionally, mass spectral data for compound 5b exerted its molecular ion peak at m/z 364.65 by intensity equals 18.87%.

(Z/E) stereochemical determination
To detect the relative configuration (E or Z) for the synthesised indole derivatives 3a&b, 4a-e and 5a-e, 2D NMR NOESY (Nuclear Over Hauser Effect Spectroscopy) experiment was performed.
Compound 4b, as a representative example, was chosen for the NOESY experiment and data obtained revealed the spatial correlation between ¼ N-NH-proton at d 7.43 ppm and azomethine -CH ¼ Nproton at d 8.35 ppm (Bond length of NOESY correlating bonds ¼ 1.7 Å in E-form), and between indole H-2 at d 8.22 ppm and azomethine proton (Bond length of NOESY correlating bonds ¼ 4.5 Å in Eform). While, no spatial interaction was observed between azomethine proton/NH or azomethine proton/indole H-2 in Z-form (Bond length of NOESY correlating bonds >6 Å in Z-form). The obtained results suggested the E-configuration for the synthesised indole derivatives.
Moreover, a theoretical method was performed to confirm the Econfiguration for the synthesised derivatives using Chem3D Ultra 12.0 and MM2 properties. Total energy, the sum of stretch, bending, stretch-bend, torsion, non-1,4 Van Der Waals, 1,4 Van Der Waals, and dipole/dipole interactions, was calculated for each Z-/E-conformer. It was noticed that the total energy of the E-form exceeded that of the Z-form for all the target compounds, (Table 1 and Figure 2).

Biological evaluation
Antimicrobial activity screening All tested compounds were subjected to antimicrobial screening against Gram-positive bacteria, Gram-negative bacteria, and yeast fungus. The obtained data revealed that the ethylthiosemicarbazide derivative, 4b, and p-methoxyphenylthiosemicarbazide compound, 4e showed selective antibacterial activity against the Gram-negative bacteria, E. coli, and Salmonella enterica. Whereas, p-tolylthiazolidinone derivative 5d exclusively exhibited bioactivity against E. coli with diameters ranging from 8 to 15 mm at 100 mg/ disc concentration compared to the standard ciprofloxacin which had inhibition zones against all tested Gram-positive and Gramnegative bacteria with diameters' range from 34 to 48 mm at a concentration of 20 mg/disc. Although our bioactive compounds showed modest antibacterial activity compared to the ciprofloxacin standard, further modifications to their structures could help increase their bioactivity or even broaden their antimicrobial spectrum of activity. On the other hand, the other nine compounds did not display antifungal or antibacterial activity, Figure 3. The result obtained was in accordance with what was reported about indole derivatives and their antimicrobial activity 4,31 . Thus, indole derivatives bearing thiazolidinone part and an aliphatic group such as Me or OEt showed the highest antimicrobial activity against both Gram-positive bacteria and Gram-negative bacteria 27 . Additionally, indole-hydrazone scaffold and methoxyphenyl substitution exhibited promising antibacterial activity, as reported by Nassar et al 31 .
Anti-oxidant activity using DPPH radical scavenging activity The in vitro antioxidant activity of semicarbazides 3a&b, thiosemicarbazides 4a-e and thiazolidinone derivatives 5a, 5c and 5e were determined by measuring the reduction capacity of DPPH radicals spectrophotometrically at 540 nm. Trolox, a water-soluble analog of vitamin E, was used as a standard. By inspecting data in Table 2, it was found that the highly anti-oxidant compound between all the tested derivatives was phenylthiazolidinone 5c with a value of 707.52 mM Trolox equivalent/mg. generally, thiosemicarbazide derivatives 4b and 4d showed high scavenging  The lowest scavenging activity on DPPH was observed in semicarbazone derivatives 3a and 3b, thiosemicarbazide derivative 4a, its thiazolidinone derivative 5a and p-methoxyphenylthiazolidinone derivative 5e with values ranging from 24.55 to 5.91 mM Trolox equivalent/mg.
It is worth mentioning that between all tested series, thiosemicarbazide moiety increased the antioxidant activity except for phenylthiazolidinone derivative 5c.

Anti-inflammatory activity
In vitro measurements of TNF-a in RAW264.7 macrophage cells The synthesised compounds were evaluated for their in vitro antiinflammatory activity using RAW264.7 macrophage cells. The excellent anti-inflammatory activity was obtained by decreasing TNF-a fold, which indicates inhibition of TNF-a production in RAW264.7 cells.
The obtained results represented in Figure 4 revealed that compounds 4d, 4e, 5b, and 5d showed the highest anti-inflammatory activity by decreasing TNF-a to reach 0.19, 0.26, 0.18, and 0.21, respectively, relative to that of indomethacin (0.22) and celecoxib (0.16). The rest of the compounds showed fold changes ranging from 0.35 to 0.53. Both the phenyl semicarbazone derivative, 3b and thiazolidinone derivative, 5a exerted the lowest activity, showing fold changes equal to 0.71 and 0.66, respectively.
From the obtained data, (Table 3), it was observed that all the tested compounds might be of good safety profile as they could   On the other hand, the p-methoxyphenylthiosemicarbazide derivative 4e and p-tolylthiazolidinone derivative 5d showed high potency towards COX-2 enzyme inhibition with IC 50 ¼ 0.81 and 0.67 lM, sequentially, compared to celecoxib (IC 50 ¼0.46 lM). While compounds 4a, 4c, 4d, 5a, 5c, and 5d exerted moderate potency towards COX-2 enzyme inhibition with IC 50 values ranging from 1.60 to 8.06 lM. The least activity was observed in ethylthiosemicarbazide derivative 4b and its thiazolidinone derivative 5b with IC 50 ¼ 86.15 and 89.22 lM, in a sequent.
Moreover, compounds containing a thiazolidinone ring with electron donating groups (CH 3/ OCH 3 ), 5d and 5e, had good COX-2 S.I values (8.59 and 9.72, in order). As a result, compounds 4e, 5d, and 5e might have a safe gastric profile more than or nearly equal to that of celecoxib.

Ulcerogenic liability
The most selective compounds, 4e and 5d, were shown to have an ulcerogenic impact when compared to the reference drug celecoxib and the ulcerogenic non-selective COX-2 inhibitor indomethacin, utilising a 50 mg/kg dosage for three consecutive days to show their gastrointestinal safety profile. The acquired findings are depicted in (Table 4). Our findings suggested that all tested compounds had a lower ulceration impact than celecoxib and indomethacin, the reference drugs, as evidenced by an ulcer index range (U.I.  Table 3. In vitro COX-1, COX-2 and 5-LOX inhibitory activity results and S.I. of the newly synthesised compounds 3a&b, 4a-e and 5a-e with indomethacin, celecoxib, and ziluton as reference drugs. ---0.58 ± 0.03 a The concentration of test compound produces 50% inhibition of COX-1, COX-2, and 5-LOX enzymes; b The in-vitro COX-2 selectivity index (IC 50 COX-1/IC 50 COX-2).   Scanning the stomach of the rats treated with the tested compounds 4e and 5d showed the following: For compound 4e: gastric tissue showed retaining of the mucous layer (black arrow), regenerated mucosal epithelial cell layers, preserved a number of parietal cells (orange arrows) and chief cells (yellow arrow), with minimal mucosal, submucosal edoema, inflammations and average muscularis mucosa (red arrow) (H & E X 200 & 400) (Figure 5(1D-3D)). In conclusion, administration of the p-methoxythiosemicarbazide derivative 4e resulted in partial loss of mucous layer with mild superficial erosions accompanied by mucosal epithelial cell layers' regeneration, preserved parietal cells and chief cells in superficial and deep mucosa, minimal mucosal, submucosal edoema, inflammations and average muscularis mucosa.
For compound 5d: gastric tissue showing minimally disrupted mucous layer (black arrow), regenerated mucosal epithelial cells layers with minimal residual surface epithelial erosions, preserved number of parietal cells (orange arrows) and chief cells (yellow arrow), with mild mucosal, submucosal edoema and inflammations and minimally decrease thickening of muscularis mucosa (red arrow) (H & E X 200) ( Figure 5(1E-3E)). In conclusion, administration of the p-tolylthiazolidinone derivative 5b resulted in partial loss of the mucous layer with mild, superficial erosions, preserved number of parietal cells and chief cells in superficial and deep mucosa, mild mucosal, submucosal edoema and inflammations and minimally decrease thickening of muscularis mucosa.

Assessment of the cardiac function biomarkers
It has been previously noted that celecoxib has cardio-toxicity in experimental rats 55 . Heart function indicators such as Troponin I, Creatine kinase-MB (CK-MB), and lactate dehydrogenase (LDH) were assessed in the current investigation to determine how the heart responded to the most active and most selective target compound, 5d. Additionally, a comparison of the assessed compound 5d to celecoxib as a reference standard was done regarding the study its histopathological changes. All of the findings were displayed in (Table 5). The findings showed that the injection of celecoxib caused a significantly higher level of the serum cardiac biomarkers Troponin I, CK-MB, and LDH when compared to normal control rats. Contrarily, animals treated with thiazolidinone derivative, 5d, had substantially lower serum levels of these indicators than rats treated with celecoxib. These findings demonstrated that, in comparison to celecoxib, compound 5d carries a considerably lower risk of cardiovascular damage.

Histopathological examination of the heart
Histopathological examination of the heart muscle of the control group shows a normal pattern of cardiac muscle fibres with normal cigar shaped nuclei (red arrows) and normally arranged blood vessels (yellow arrows) (H & E X 200 & X 400) ( Figure 6(1A and  2A)). On the contrary, the heart section obtained from celecoxib group showed markedly inflamed cardiac muscle fibres with vacuolar degenerated myocytes (yellow arrow), edoema and dilated ectatic blood vessels containing plasma fluid (red arrow) (H & E X 200) (Figure 6(1B)); another view revealed markedly inflamed cardiac muscle fibres with vacuolar degenerated myocytes (yellow arrow), degenerated myocytes with brown pigmentation (black arrow) edoema, inflammatory cells (orange arrow) and dilated ectatic blood vessels containing plasma fluid (red arrow) (H & E X 400) (Figure 6(2B)). In conclusion, prolonged administration of celecoxib resulted in scattered inflamed cardiac muscle fibres with vacuolar degenerated myocytes with brown pigmentation, edoema as well as markedly congested and dilated ectatic blood vessels containing plasma fluid in the heart muscle. Alternatively, cardiac histopathological examination of the thiazolidinone derivative 5d treated group revealed little or no toxic effects on the heart and showed viable cardiac muscle fibres retained to their normal pattern with regenerated myocytes and normal arranged blood vessels (yellow arrows) (H & E X 200) (Figure 6(1C)); another view revealed cardiac muscle fibres retained to their normal pattern with regenerated myocytes, normal cigar shape nuclei (red arrow) and normal arranged blood vessels (yellow arrows) (H & E X 400) (Figure 6(2C)).

Molecular docking study
In this study, the mode of action of N-methylsulfonyl indole derivatives 3a&b, 4a-e, and 5a-e as novel COX-2, and 5-LOX inhibitors were determined via molecular docking studies. Docking of COX-2 ligand, celecoxib, and 5-LOX ligand, arachidonic acid, inside the binding site of COX-2 (Protein Data Bank code: 3ln1) and 5-LOX (Protein Data Bank code: 3V99) was performed to compare between the key structural features for both ligands and the designed candidates. The docking energy affinity (Kcal/mol), interactions and amino acid residues indicated the binding mode for the developed new compounds such as COX-2 and/or 5-LOX inhibitors.
Regarding the COX-2 docking study, the docking study of celecoxib, a selective COX-2 inhibitor, showed a binding energy of À7.52 Kcal/Mol. It formed three hydrogen bonding interactions with Gln178, Ser339 and Arg499 amino acid residues with NH 2 and SO 2 groups and one arene-H interaction with P-SO 2 NH 2 Ph moiety, (Figure 7, Table 6).
Additionally, docking studies for testing derivatives revealed that the energy of binding interactions ranged from À6.44 to À2.40 Kcal/Mol, and formed one to five binding interactions or without any interactions at all. The most active COX-2 inhibitors, p-methoxyphenylthiosemicarbazide derivative, 4e and p-tolylthiazolidinone derivative, 5d showed the highest energy scores of À6.37 and À6.44 Kcal/Mol and formed five and one binding interaction(s), respectively, with Ser516, Phe504, His75 and Ser339 amino acid residues with indole ring (phenyl and pyrrole), C ¼ S and SO 2 CH 3 pharmacophores, in addition to a p-tolyl moiety in compound 5d, (Figures 8 and 9, Table 6).
On the other hand, the last two derivatives, as COX-2 inhibitors, were ethyl derivatives of thiosemicarbazide, 4b, and thiazolidinone, 5b. They revealed binding energy scores of À2.62 and À2.4026, sequentially. Compound 4b didn't form any type of binding interaction, while compound 5b exerted one arene-hydrogen interaction between the pyrrole ring of indole and Ser339 amino acid residue, (Table 6). Concerning docking studies inside the 5-LOX enzyme, data obtained revealed that the active site of the enzyme formed two hydrogen bonding interactions between His432 and His600 amino acid residues and a -(-COO -) group of arachidonic acid, the redocked co-crystallised ligand compound with a binding energy score of À7.82 Kcal/Mol ( Figure 10, Table 6).
The most active p-tolyl thiosemicarbazide derivative as a 5-LOX inhibitor, 5d, had the highest binding energy score of À9.91 Kcal/ Mol, higher than that of the ligand. It had two hydrophobic interactions between the phenyl of the indole ring and His432 and His600 amino acid residues (the same as that of the ligand) ( Figure 11, Table 6).
Moreover, most of the tested derivatives showed energy binding interactions nearly equal to the ligand from À7.78 to À7.19 Kcal/Mol and formed binding interactions of one to two with Gln363, Asn554, His600, His432, His367, and His372 amino acid residues, ( Table 6).
On the other hand, semicarbazide derivatives 3a&b, ethylthiosemicarbazide derivative 4b, p-tolylthiosemicarbazide 4d, and ethyl or phenyl thiazolidinone derivatives 5b&c showed energy binding interactions between À6.96 to À6.03 Kcal/mol, formed one or two binding interaction(s) with His367, Gln557, His432, and Lys 409 amino acid residues or without any binding interactions as in 3a, 4b, and 5b, ( Table 6). In conclusion, the molecular docking study results of COX-2 and 5-LOX enzymes indicated that the p-methoxy derivative of thiosemicarbazide 4e had COX-2 binding activity, while the p-tolyl derivative of thiazolidinone 5d, showed dual COX-2/5-LOX binding affinities. Both of them contain an electron-donating group in the para position of the phenyl ring.

Molecular properties and drug-likeness
Lipiniski's rule of five (RO5) analysis was used in this study to predict the biochemical properties of the synthesised target compounds. All compounds achieved Lipinisk's rule of five standard values with a violation number of zero, (Table 7). Thus, the molecular weight of the target derivatives ranged from 280-442 g/Mol. That means less than the standard value of 500 g/Mol. Also, both hydrogen-bond acceptor and donor of tested compounds were in the range of 4 À 7 and 0-3, respectively, they didn't exceed the acceptable values (No. HBA <10 and No. HBD <5). Moreover, the number of rotatable bonds, which indicate molecular flexibility, was from 3 to 5 (standard value <10). Lipophilic indicator LogP (octanol/water) partition co-efficient was evaluated for the target compounds. All showed high to moderate membrane permeability with partition co-  Å. This was achieved by the target compounds (75 À 106 Å). On the other hand, druglikeness scores were acceptable for the target compounds, especially those containing semicarbazone moiety 3a (-0.07) and for p-methoxyphenyl thiosemicarbazide derivative 4e (-0.04).

Bioactivity prediction
Interaction between synthesised compounds and certain drug targets might be a tool for their bioactivity prediction. From these drug targets, a G-protein coupled receptor (GPCR), an ion channel modulator, a kinase inhibitor, a nuclear receptor ligand, and protease inhibitor were studied. To consider the activity of the synthesised compounds, they should possess values >0.00 to be good lead structure, À0.50 to 0.00 as moderately bioactive, or < À0.50 to be inactive.
By inspecting the results obtained (Table 8), it was found that semicarbazone derivatives 3a and 3b showed good lead structures for G-protein coupled receptor (GPCR), while the rest of the compounds had moderate bioactivity.
All tested derivatives were inactive as ion channel modulators and protease inhibitors. Compounds 3b, 4c, and 4e bearing phenyl semicarbazone/thiosemicarbazone or p-methoxyphenylthiosemicarbazone scaffolds were moderately active as kinase inhibitors and nuclear receptor ligand, in addition to semicarbazone derivative 3a and p-tolylthiosemicarbazone derivative 4d that might be considered as kinase inhibitors.
In silico ADME prediction Pharmacokinetic properties (absorption, distribution, metabolism and excretion) for the synthesised compounds were predicted using in silico ADME prediction.
The obtained results were recorded in Table 9. All the target compounds showed high intestinal absorption with values    Table 9. ADME prediction for the synthesised indole derivatives 3a&b, 4a-e and 5a-e.

HIA (%)
In vitro Caco-2 cell permeability (nm/sec) ranging from 93.40 to 99.66% and reached 100% in the case of thiazolidinone derivative 5e. Low to moderate permeability results were observed for in vitro CaCo-2 and MDCK cells in the range 0.58-24.16 and 0.04-19.81 nm/sec, respectively. Most of the compounds exerted a strong binding effect on plasma proteins higher than 90% except semicarbazone derivative 3a and thiosemicarbazone derivatives 4a and 4b. They possessed plasma binding values of 57.14, 79.56, and 86.83%, sequentially. Moreover, all the synthesised compounds exerted low absorption into the CNS. They couldn't penetrate it. They had values in the range of 0.01-0.21 (i.e. <0.40, the standard value). Consequently, all the synthesised targets might be a good lead for the transdermal delivery systems. They showed maximum skin permeability with logKp values ranging from À2.88 to À1.74 cm/h.

Metabolism prediction
Metabolism prediction was obtained by studying phase I metabolism parameters. Data in Table 10 shows that most of the compounds could inhibit the cytochrome P450 isoform CYP-2C9. While none of them could inhibit other cytochrome isoforms such as CYP-2C19, CYP-2D6 and CYP-3A4.

Conclusion
A novel series of N-methylsulfonyl indole derivatives fused with semicarbazone 3a&b, thiosemicarbazone 4a-e, and thiazolidinone 5a-e scaffolds were synthesised and evaluated for their biological activities. The antimicrobial screening revealed that thiosemicarbazide derivatives, 4b and 4e showed selective antibacterial activity against the Gram-negative bacteria, E. coli and Salmonella enterica. Whereas thiazolidinone derivative 5d exhibited bioactivity against E. coli only. On the other hand, the tested compounds did not display antifungal activity. The in vitro antioxidant activity of the target compounds showed that phenylthiazolidinone derivative 5c had the highest anti-oxidant activity among all the tested derivatives with a value of 707.5 mM Trolox equivalent/mg. For most thiosemicarbazide derivatives, their anti-oxidant activity was higher than their thiazolidinone analogs. The lowest scavenging activity on DPPH was observed in semicarbazone derivatives 3a and 3b, unsubstituted thiosemicarbazide derivative 4a, its thiazolidinone analog 5a and p-methoxyphenylthiazolidinone derivative 5e. The in vitro anti-inflammatory results by measuring TNF-a in RAW264.7 macrophage cells revealed that compounds 4d, 4e, 5b and 5d had the highest anti-inflammatory activity, while, phenyl semicarbazone derivative, 3b and thiazolidinone derivative, 5a exerted the lowest activity. Furthermore, the synthesised compounds were tested against COX-1, COX-2 and 5-LOX enzymes. Compound 4e could be classified as a selective COX-2 inhibitor and compound 5d had dual COX-2/5-LOX inhibitory activity. As a result, measuring cardiac biomarkers (LDH, CK-MB and Tn-I) for compound 5d elicited low levels. A histopathological study affirmed the cardioprotective profile for the target compound 5d. Additionally, for both derivatives, 4e and 5d, ulcerogenic liability and the histopathological study confirmed their safety effect on the gastric mucosa. Moreover, a docking study for the target compounds inside COX-2 and 5-LOX active sites was performed to explain their plausible binding mode. Finally, an ADMET study and pharmacokinetic properties were applied and proved the promising activity of the new compounds. In conclusion, the newly developed compounds represent biologically active multi-target candidates with fewer side effects. Structure-activity relationship (SAR) is represented in the following Figure 12.